Our goal is to identify and characterize normal prostate epithelial stem cells, to determine whether they are targets of malignant transformation and to define their relationship to prostate cancer stem cells. We have identified in vivo a slow-cycling population of cells with high proliferative potential in the proximal region of murine prostatic ducts. These cells express high levels of stem cell antigen 1 (Sca-1) and contain a subset of basal cells expressing ?4 integrin and a smaller subset coexpressing both ?4 integrin and prominin, (CD133), a protein also expressed by primitive human prostate cells. We find that a small fraction of Sca-1high cells express high levels of aldehyde dehydrogenase (ALDH), an enzyme highly expressed in other stem cells. We conclude that the cells within the prostate gland differ markedly in their cycling status, their proliferative potential and their surface markers, providing an opportunity for further stratification into more homogeneous subsets. We propose that the stem cell-containing subset is the target of malignant transformation and that, similar to the normal prostate, prostate tumors contain a population of self-renewing cancer stem cells responsible for propagating the tumor. These hypotheses will be tested in three aims that exploit our ability to isolate viable subsets of Sca-1high cells and to test their proliferative potential in vitro and in vivo. In Aim 1, Sca- 1high cells will be fractionated into subpopulations expressing ?4 integrin, prominin, ALDH and other proteins we have already identified or that we will seek through additional microarray analysis. The biological role of one such protein, ALDH, in prostate stem cell biology will also be explored. Our multipronged approach using in vitro and in vivo assays for determining proliferative and differentiative potential should identify the phenotype of cells with the highest in vivo prostate-regenerating potential. The microarray analysis of subsets with increasing regenerative potential will be used to define a prostate stem cell gene expression signature. In Aim 2 we will determine if stem cells are specific targets of malignant transformation. Subsets of Sca-1high cells will be infected with lentivirus containing activated AKT1 or c-Myc, two important contributors to human prostate cancer, and the transformed populations will be characterized. In Aim 3 we will identify cancer stem cells within prostate tumors originating from the targeted deletion of Pten. We showed that the Sca-1high fraction of tumor cells contains tumor-initiating cells. We will use additional markers to further purify these cells that will be identified by their tumorigenicity and malignant properties. The stem cell gene expression signatures of normal and cancer stem cells will also be compared. The successful completion of these aims will yield new insights into the initiation and maintenance of experimentally induced prostate cancer and pave the way to a better understanding of the etiology of prostate carcinoma and benign prostatic hypertrophy, as both diseases are considered to arise from the aberrant proliferation of stem cells.